Opioid receptors undergo axonal flow.

Previous studies have indicated the presence of opiate receptors on axons of the rat vagus nerve and on other small diameter fibers. In examinations of the effect of ligation on the distribution of receptors in the vagus nerve by in vitro labeling light microscopic autoradiography, a large buildup of receptors was found proximal to the ligature. This result indicates an axonal flow of receptors.

Alterations in cortical muscarinic receptors following cholinotoxin (AF64A) lesion of the rat nucleus basalis magnocellularis.

Cortical choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), muscarinic receptors and sodium-dependent, high-affinity, choline uptake (SDHACU) sites were examined in the rat brain following unilateral stereotaxic injection of the cholinotoxin, AF64A, into the nucleus basalis magnocellularis (NBM). Injection of AF64A resulted in a significant loss of presynaptic cholinergic markers in the cortex without alteration in TH and TPH activity. The binding to SDHACU sites was reduced to background values in the NBM and increased in the central amygdala (Ce) and cortex. The increase in cortical [3H]QNB binding was the result of a change in muscarinic receptor number (BMAX) and not a change in receptor affinity (KD). Examination of muscarinic receptor subtypes demonstrated a reduction of M1 receptor binding in the cortex and NBM without any alteration in the Ce. Non-M1 binding was significantly increased in all the laminae of the cortex and in the Ce, but decreased in the NBM. These data suggest that there exists a population of M1 receptors on NBM projections to the cortex and that NBM projections influence a population of postsynaptic receptors in the cortex and Ce which are not of the M1 subtype.

Transport of receptors.

The axonal transport of neurotransmitter receptors is thought to be a common phenomenon in many neuronal systems. The "machinery" for receptor (protein) "assembly" is found in the cell bodies of neurons and the "manufacture" of receptors takes place there. These receptors are then "shipped" to their ultimate destinations by a transport process. This is an axonal transport mechanism in the case of presynaptic receptors. Some form of transport process may also exist to send receptors out into the dendritic arborizations of neurons, although the latter is more difficult to verify. Axonal transport has been demonstrated, in the peripheral nervous system, for many different neurotransmitter receptors. In the central nervous system, the results are less clear, but indicate the presence of a transport mechanism for catecholamine, acetylcholine, and opiate sites. One important component then, in the development of receptors, is the transportation to terminal membrane sites where they are ultimately incorporated and available for interaction with neurotransmitters and drugs.

Characterization of the binding and comparison of the distribution of benzodiazepine receptors labeled with [3H]diazepam and [3H]alprazolam.

The binding characteristics of [3H]diazepam and [3H]alprazolam were obtained by in vitro analysis of sections of rat brain. Dissociation, association, and saturation analyses were performed to optimize the conditions for obtaining selective labeling of benzodiazepine receptors with the two tritiated compounds. Both drugs approached equilibrium rapidly in vitro. Rosenthal analysis (Scatchard plot) of the saturation data indicated a similar finite number of receptors was being occupied by both ligands. Competition studies, using various ligands to inhibit both [3H]diazepam and [3H]alprazolam indicated that these two compounds bind to the tissue sections as typical benzodiazepine drugs and apparently do not overlap onto other subtypes of receptors. These experiments were performed by both binding assay in tissue sections and by light microscopic autoradiography. The major difference between the labeling of the two compounds is represented by the peripheral benzodiazepine sites, which are recognized by [3H]diazepam, but not occupied by [3H]alprazolam (at nanomolar concentrations). This difference was readily apparent in the autoradiograms. Other pharmacokinetic or pharmacodynamic properties must distinguish these two benzodiazepines.

Distribution of [125I]angiotensin II binding sites in the rat brain: a quantitative autoradiographic study.

Angiotensin II receptors have been localized by quantitative autoradiography in the rat central nervous system after labeling with [125I]angiotensin II. A highly discrete distribution of these receptors was found throughout the rat brain. The highest density was seen in regions of the medulla, hypothalamus and circumventricular organs where angiotensin II could potentially produce cardiovascular, dipsogenic and neuroendocrine responses. The distribution of angiotensin II receptors correlates relatively well with the previously reported distribution of angiotensin immunoreactive nerve terminals as well as areas determined by various physiological techniques to be sensitive to angiotensin II. Finally, the anatomical localization of angiotensin II receptor populations has revealed several areas of the brain where the effects of this peptide have not been investigated. Many of these nuclei are involved in the transmission and processing of somatic and visceral sensory information. These results suggest a broader role for the central renin-angiotensin system in modulating several types of sensory input.

The D1 dopamine receptor in the rat brain: quantitative autoradiographic localization using an iodinated ligand.

The distribution of dopamine D1 receptors in the rat, labeled with [125I]SCH 23982, was studied using a quantitative in-vitro light-microscopic autoradiographic method. The binding of [125I]SCH 23982 to slide-mounted tissue sections and membrane preparations of prefrontal cortex was saturable, specific and of high affinity. Scatchard analysis revealed a Kd of 1.15 +/- 0.47 nM and Bmax of 8.76 +/- 0.34 fmol/mg tissue in prefrontal cortex membranes and a Kd of 1.27 +/- 0.14 nM and Bmax of 67.6 +/- 3.75 fmol/mg tissue in slide-mounted tissue sections at the level of the striatum. [125I]SCH 23982 was found to predominantly label D1 receptors, but a small fraction of the binding was to serotonin receptors. D1 receptors were found throughout the forebrain and were concentrated in the substantia nigra pars reticulata, accumbens nucleus, caudate putamen, entopeduncular nucleus, olfactory tubercle and the major island of Calleja. [125I]SCH 23982 binding to serotonin receptors was concentrated in the cortices, dorsal raphe, central gray, anterior hypothalamic area and the molecular cell layer of the cerebellum. Knowledge of the distribution of D1 receptors may increase our understanding of the role of D1 receptors in central nervous system dopaminergic function. Furthermore, data on the potential sites of interaction of [125I]SCH 23982 with serotonin receptors may help to understand the complex physiology and pharmacology of the primarily D1 selective compound.

Methamphetamine-induced reduction in D1 and D2 dopamine receptors as evidenced by autoradiography: comparison with tyrosine hydroxylase activity.

As determined by autoradiographic techniques, multiple high doses of methamphetamine elicited a reduction in dopamine receptor population (both D1 and D2) in several areas of the rat central nervous system. D1 receptors were labeled with the D1-selective antagonist, [3H]SCH 23390, and D2 receptors were labeled with the D2-selective neuroleptic, [3H]sulpiride. Scatchard analysis, obtained from saturation data in caudate-putamen, indicated that the receptor alterations were due to a decrease in the number of receptors (Bmax) without an apparent change in affinity (Kd). A time course demonstrated that five doses of methamphetamine were required to elicit significant changes in receptors in most brain areas examined. The onset of the receptor alterations in various brain regions correlated with the development of methamphetamine-induced depression of striatal tyrosine hydroxylase activity. In most brain areas, the dopamine receptors returned to normal within 7 days following methamphetamine.

Distribution of opiate receptors in the monkey brain: an autoradiographic study.

By employing both in vivo and in vitro labeling techniques, opiate receptors were labeled with tritiated diprenorphine in the monkey brain and localized by light microscopic autoradiography. Both methods of labeling gave similar results, allowing a description of discrete areas having opiate binding sites. High concentrations of opiate receptors were found in the substantia gelatinosa of the spinal cord, nucleus tractus solitarius, area postrema, lateral parabrachial nucleus, substantia grisea centralis, several nuclei of the thalamus and hypothalamus, substantia innominata and in the amygdala. In the monkey pituitary, receptors were found in the neurohypophysis. These results correlate well with those found in autoradiographic studies of the rat brain although there are a few notable differences. Many of the opiate receptor distributions can be correlated with anatomical loci of brain functions known to be influenced by administration of opiate compounds.

Autoradiographic determination of neurotransmitter receptor distributions in the cerebral and cerebellar cortices.

Light microscopic autoradiographical localization of drug and neurotransmitter receptors provides investigators with a tool to determine the sites of drug action with a high degree of anatomical resolution. In the cortex, where there is a distinct laminar organization, the differential distribution of receptors can be determined. We have found that differences in the density of specific receptors exist between layers of the cerebral cortex. Our observations include the localization of muscarinic cholinergic, benzodiazepine, opiate, neurotensin, histamine-H1, alpha-adrenergic, beta-adrenergic, and high-affinity GABA receptors in the cerebral cortex. Of this list of neurotransmitters only significant concentrations of benzodiazepine, GABA, and beta-adrenergic receptors were found in the cerebellar cortex. Receptor localizations such as these can be coupled with information from immunohistochemical studies to provide the basis for future experimentation in order to define neurotransmitter-specific pathways in the cerebral and cerebellar cortices.

Relative affinity of quazepam for type-1 benzodiazepine receptors in brain.

Quazepam and its major metabolite, 2-oxoquazepam, were the first benzodiazepine (BZ) compounds shown to be capable of differentiating between central nervous system (CNS) subtypes of BZ receptors. Initial studies suggested that quazepam preferentially binds to the BZ1-receptor population with high affinity at relatively low doses. Subsequent binding experiments, performed in both human and rodent brain, confirmed this hypothesis for both the parent compound and its principal metabolite. Autoradiographic studies revealed a differential distribution of BZ1 and BZ2 receptors within regions of the brain associated with behavior, sensory input, control of movement, and coordination. BZ1 receptors are concentrated in specific, well-localized regions of the brain. BZ2 receptors are more widespread and diffusely localized throughout most brain regions. These observations suggest that a selective BZ1 agent such as quazepam might have a different pharmacologic profile than other nonselective benzodiazepines. Restricting the number of sites where BZ drugs act has been proposed as a strategy for producing a more circumscribed therapeutic effect; the BZ1 selectivity of quazepam embodies this concept.

Receptor alterations associated with serotonergic agents: an autoradiographic analysis.

Controversy exists concerning whether receptor down-regulation is involved in the efficacy of antidepressants. Many investigators believe that norepinephrine (NE) receptor down-regulation is more important than serotonin (5-HT) receptor down-regulation. The ability to accurately determine which receptor types or subtypes have been down-regulated has been impaired by the lack of sufficiently specific ligands for labeling these receptor subtypes. Studies that have attempted to examine 5-HT2 receptor down-regulation have used [3H]-ketanserin as the ligand of choice to label 5-HT2 receptors, but this ligand also labels a nondescript site. The binding of [3H]-ketanserin to sites other than 5-HT2 receptors can be examined and controlled for by autoradiographic techniques. The authors briefly review potential problems involved in analyzing receptor binding after antidepressant treatment and present new findings of receptor alterations in rat brain as examined by autoradiographic techniques following chronic exposure to fluoxetine (a selective 5-HT uptake inhibitor that has been shown to be an effective antidepressant). Laboratory animals injected with fluoxetine showed receptor down-regulation (reduced density) in the serotonergic system. A provocative and potentially important finding of this study is that this selective 5-HT uptake blocker also down-regulates beta-adrenergic receptors in the CNS.

Time dependent changes in DA uptake sites, D1 and D2 receptor binding and mRNA after 6-OHDA lesions of the medial forebrain bundle in the rat brain.

Quantitative receptor autoradiography and in situ hybridization techniques were used to examine the temporal pattern of changes in dopamine uptake sites, D1 and D2 receptors and their transcripts in the striata of animals lesioned with 6-hydroxydopamine. Animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4, 6, 8, and 16 weeks postlesion. Degeneration of the nigrostriatal pathway induced a significant loss of dopamine uptake sites in the ipsilateral caudate putamen of all lesioned animals. D1 receptor binding was significantly increased in the caudate putamen on the lesioned side from 1 week to 16 weeks postlesion, whereas the expression of D1 receptor mRNA did not show any change during this period. There was a significant upregulation of D2 receptor binding as well as D2 mRNA from 2 weeks to 8 weeks postlesion. However, at 16 weeks postlesion, D2 receptor binding continued to increase, whereas the mRNA appeared to compensate. These studies show that a different regulatory mechanism may exist between these two DA receptor subtypes. D1 receptor changes occur at the post-transcriptional or translational level, whereas D2 alterations occur by both transcriptional and translational processes. These studies also indicate that the postsynaptic supersensitivity observed in D1 receptors may not be accompanied by a corresponding increase in D1 receptor mRNA.

GABAA receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system.

The regional distribution of radioactive ligand binding in rat brain for the different receptors of the gamma-aminobutyric acidA (GABAA)-benzodiazepine receptor/chloride channel complex was measured on tissue sections by autoradiography. Seven ligands were employed including [3H]muscimol for high-affinity GABA agonist sites; [3H]bicuculline methochloride and [3H]SR-95531 for the low-affinity GABA sites; [3H]flunitrazepam for benzodiazepine sites, and [3H]2-oxo-quazepam for the 'BZ1'-type subpopulation; and [35S]t-butyl bicyclophosphorothionate (TBPS) and [3H]t-butyl bicyclo-orthobenzoate (TBOB) for convulsant sites associated with the chloride channel. Allosteric interactions of benzodiazepine receptor ligands with [35S]TBPS binding also were examined in membrane homogenates. Comparison of 19 brain regions indicated areas of overlap between these ligands, but also significant lack of correspondence in some regions between any two ligands compared. In particular, the cerebellum, thalamus, hippocampus, substantia nigra and superior colliculus showed enrichment in the binding of some ligands compared to others, and other brain regions showed smaller discrepancies. In addition to the previously observed discrepancies between high-affinity GABA agonists binding and benzodiazepine receptor distribution, especially in the cerebellum, and the well-documented differences in 'BZ1'-selective versus non-selective ligands, significant differences were observed in comparing GABA agonists with antagonists, one antagonist with another, GABA ligands with benzodiazepine or convulsant sites, and even between the two convulsants TBPS and TBOB. The major factor in regional variations within one ligand and between ligands involves differences in binding site densities, although other factors such as endogenous ligands and conformational flexibility may contribute to these findings. The lack of correspondence between components of the GABAA-receptor complex is most consistent with the existence of subtypes that vary in their binding affinities or even binding capabilities. At least four such subtypes are required to explain the regional dissimilarities between ligands. It is likely that these subtypes based on binding alone correspond to different gene products demonstrated recently by molecular cloning and protein chemistry, indicating a pharmacological heterogeneity that might be exploited with subtype-specific drugs showing desirable clinical profiles.

Selective, unilateral cortical infarction increases striatal muscarinic receptor binding: potential evidence for cortical modulation of intrastriatal cholinergic transmission.

The effects of unilateral cortical infarction on subcortical (striatal) muscarinic receptors in rat brain were studied by means of in vitro receptor autoradiography using [3H]quinuclidinyl benzylate ([3H]QNB) and [3H]pirenzepine. The cortical lesions could be produced without compromising subcortical structures. A dramatic (20 to 59 per cent) increase in striatal [3H]QNB binding was observed ipsilateral to the damaged cortex. The increase in binding was greatest in the caudate-putamen, but was also noted in the nucleus accumbens. [3H]Pirenzepine binding (labeling M1 receptors) was also increased but to a lesser degree, as was [3H]QNB binding in the presence of 100 nM (unlabeled) pirenzepine (an indirect means of labeling M2 receptors). The results show that unilateral cortical infarction results in an upregulation of striatal muscarinic receptors, and suggest that both the M1 and M2 subtypes contribute to this effect. These findings provide evidence for cortical modulation of intrastriatal cholinergic transmission.

Comparison of the distribution of D-1 and D-2 dopamine receptors in the rat brain.

The distribution of dopamine type 1 (D-1) and dopamine type 2 (D-2) receptors in the brain have been compared as assessed by the technique of autoradiography after labelling with highly selective ligands. D-1 receptors, as evidenced by the specific binding of [3H]R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-IH-3-benzazepine -7- ol (SCH 23390), were found in high concentrations in the caudate-putamen, nucleus accumbens, islands of Calleja, olfactory tubercle and the zona reticulata of the substantia nigra. A similar but distinct distribution was seen for [3H]sulpiride, a ligand which is highly selective for D-2 receptors. Like [3H]SCH 23390, this ligand also labelled the caudate-putamen, nucleus accumbens, islands of Calleja and the olfactory tubercle; however, only a very low density of D-2 receptors could be found in the zona reticulata of the substantia nigra, while a greater degree of binding was present in the zona compacta. Additional brain areas which contained D-1 but not D-2 receptors included the cerebral cortex, accessory olfactory nucleus, amygdala, thalamus, suprachiasmatic nucleus, choroid plexus, claustrum, endopiriform nucleus, zona incerta, dorsal lateral geniculate nucleus and the dentate gyrus. D-2 receptors were also found in areas which appeared to contain only low amounts of D-1 receptors such as the glomerular layer of the olfactory bulb, bed nucleus of the stria terminalis, hypothalamus, habenula, stratum lacunosum moleculare of the hippocampus, intermediate lobe of the pituitary, lateral mammillary nucleus, periaqueductal gray, inferior colliculus, nodulus of the cerebellum and the dorsal horn of the spinal cord. The results show the precise localization of dopamine receptors throughout the brain and provide a means of direct comparison between the distribution of dopamine receptor subtypes. These subtypes are pharmacologically and anatomically distinct entities and their comparison indicates areas where additional biochemical and neuroanatomical studies may be performed to elucidate the roles for these receptor subtypes in the central nervous system.

D1-receptor antagonists: comparison of [3H]SCH39166 to [3H]SCH23390.

A radiolabeled form of the benzonaphthazephine, SCH39166 was used to characterize the binding of this D1 antagonist in cortex, and an autoradiographic comparison of the localization of [3H]SCH39166 to [3H]SCH23390 (D1 antagonist and forerunner of SCH39166) binding was performed. The Kd for [3H]SCH39166, calculated from dissociation and association rate constants (1.09 nM), was comparable to the Kd value derived from Scatchard analyses of saturation data (1.74 nM). [3H]SCH39166 binds to brain tissue in a saturable manner with high affinity and low non-specific binding. Inhibition of [3H]SCH39166 binding by dopaminergic and serotonergic agents supports the hypothesis that this is indeed a D1-specific compound with little overlap onto serotonin (5-HT) receptors. The affinity of [3H]SCH39166 for 5-HT2 and 5-HT1c receptors is at least an order of magnitude lower than the affinity of [3H]SCH23390 for these same receptor sites. Quantitative autoradiographic analysis of [3H]SCH39166 and [3H]SCH23390 binding indicates high D1-receptor density in the caudate-putamen, nucleus accumbens, olfactory tubercle, substantia nigra and entopeduncular nucleus. Low levels of binding (not significantly above background) were detected with [3H]SCH39166 in lamina IV of the cortex and in choroid plexus; areas which had significant [3H]SCH23390 binding and are known to have a high density of 5-HT (5-HT2 and 5-HT1c respectively) receptors.

In vivo autoradiography of [3H]SCH 39166 in rat brain: selective displacement by D1/D5 antagonists.

The purpose of this study was to examine the receptor occupancy of D1/D5 antagonists for D1-like dopamine receptors in rat brain using [3H]SCH 39166, a highly selective D1/D5 antagonist with low affinity for 5HT2 receptors. A single concentration of triated SCH 39166 was administered to rats, with or without competing doses of the Dl/D5 antagonist SCH 23390 and unlabeled SCH 39166. the D2-like antagonists haloperidol or the 5-HT, antagonist ketanserin. The bound radioactivity in the cortex, striatum, nucleus accumbens and olfactory tubercle was then quantified using an in vivo autoradiographic procedure. The results indicated that [3H]SCH 39166 was dose dependently displaced by the Dl/D5 antagonists in regions associated with both the nigro-striatal pathway and the mesolimbic dopamine pathway, particularly the nucleus accumbens. Neither haloperidol nor ketanserin displaced [3H]SCH 39166 in any of the regions examined. The data were compared with previously published data examining the in vivo binding of [3H]SCH 39166 in rat brain homogenates. The relative values obtained were comparable to values detected in rat brain homogenates after in vivo binding of [3H]SCH 39166.

Decreased beta-adrenergic receptors in rat brain after chronic administration of the selective serotonin uptake inhibitor fluoxetine.

Fluoxetine, a novel antidepressant compound that potently and selectively inhibits serotonin uptake, was chronically administered to laboratory rats. Using in vitro receptor autoradiographic techniques, we found that the binding of [3H]-dihydroalprenolol [( 3H]-DHA) decreased significantly in frontal cortex layers. Analysis of saturation experiments indicated that the reduction was due to a change in number but not affinity of [3H]-DHA binding sites. The data support the hypothesis that the mechanism of action of most antidepressant compounds involves a change in beta-adrenergic receptor function.

Dopamine receptors in the rat brain: quantitative autoradiographic localization using [(3)H]sulpiride.

In vitro labeling of tissue sections with [(3)H]sulpiride has been utilized in the present study to autoradiographically localize D(2)-dopamine receptors in the rat brain. Preliminary biochemical studies, using slide-mounted tissue sections, were performed to define the optimal labeling conditions for this binding. Autoradiograms were generated by apposition of the labeled tissue sections to tritium-sensitive film. Specific binding sites for [(3)H]sulpiride were localized to the caudate-putamen, nucleus accumbens, olfactory tubercle, glomerular layer of the olfactory bulb, pituitary, laminae I and III of the entorhinal cortex, substantia nigra, lateral mammillary nucleus and the stratum-lacunosum moleculare of the hippocampus. The high selectivity of [(3)H]sulpiride for the D(2)-dopamine receptor indicates that it is a valuable tool for the autoradiographic localization and quantitation of neuroleptic receptors.

CNS distribution of D1 receptors: use of a new specific D1 receptor antagonist, [3H]SCH39166.

D1 dopamine receptors have been localized using a radioactive form of a new specific antagonist, [3H]SCH39166. This compound has been shown, in in vitro binding studies, to be highly selective for the D1 receptor subtype; more so than its predecessor, [3H]SCH23390. These ligand binds saturably, reversibly and with high affinity. Use of appropriate conditions produces a high signal to noise binding ratio to D1 receptors in slide-mounted tissue sections. Autoradiographic localization of radiolabeled receptors shows high densities of the D1 receptor subtype in such brain structures as the caudate-putamen, nucleus accumbens, entopeduncular nucleus, and the substantia nigra pars reticulata. A lower density of receptors is found in a few other areas including lamina VI of the cerebral cortex. A distinct paucity of binding was apparent in lamina IV of the cerebral cortex and in the choroid plexus, two areas thought to have D1 receptors. SCH39166 thus represents a superior ligand for obtaining selective labeling of D1 receptors in autoradiographic and binding studies.